Figure 2

Analysis of the senescence associated secretory phenotype (SASP) of melanoma cells treated with a DNA damaging agent. (a) Conditioned media was obtained from shScr cells exposed to TMZ or DMSO, and quantitative analysis of the proteins identified in both conditions using the shotgun approach was performed. The Venn diagram shows proteins statistically exclusively detected in shScr DMSO (non-senescent) or shScr TMZ (senescent) cells (P < 0.05). The tables show the 10 top proteins exclusively found in each condition (complete list in Supplementary Dataset S3A). Selected differential proteins discussed in the text are highlighted in bold. (b) Volcano plot showing proteins with differential abundance between conditions. Each dot represents a protein identified in at least four of the six biological replicates, red and blue dots represent proteins significantly increased or decreased, respectively, in shScr TMZ cells vs shScr DMSO cells (BH q-value < 0.05) (the identity of each dot is shown in Supplementary Dataset S3A). Selected differential proteins discussed in the text are labeled. (c) Pathway enrichment analysis with the Reactome database, considering the proteins present exclusively in senescent cells (shScr TMZ) or significantly increased in senescent (shScr TMZ) vs non senescent cells (shScr DMSO) (complete list in Supplementary Dataset S3B). The relation between the number of proteins from the sample and the number of proteins found in the term is shown in brackets. (d) Mice were injected subcutaneously with 2.5 × 105 cells in the right flank. On days 10, 12 and 14 p.t.i. conditioned media (100 μl) from shScr DMSO, shMfn1 DMSO, shScr TMZ or shMfn1 TMZ was injected intratumorally (arrows), and tumor size was measured. Results show the mean ± SE (n = 23–25, per group). Two-way ANOVA and Tukey post-hoc for multiple comparisons *P < 0.05, with respect to shScr DMSO, #P < 0.05, with respect to shMfn1 TMZ. (e) Survival curves for the animals treated as described in (d). Log-rank test, curve comparison *P < 0.05, (n = 23–25).